Viruses are by far the most abundant parasites on earth, and they have been found to infect all types of cellular life including animals, plants, and bacteria. However, different types of viruses can infect only a limited range of hosts and many are species-specific. Some, such as smallpox virus for example, can infect only one species—in this case humans, and are said to have a narrow host range. Other viruses, such as rabies virus, can infect different species of mammals and are said to have a broad range. The viruses that infect plants are harmless to animals, and most viruses that infect other animals are harmless to humans. Examples of common human diseases caused by viruses include the common cold, influenza, chickenpox and cold sores. Many serious diseases such as ebola, AIDS, avian influenza and SARS are caused by viruses. The relative ability of viruses to cause disease is described in terms of virulence. A pandemic is a worldwide epidemic. The 1918 flu pandemic, for example, commonly referred to as the Spanish flu, was a category 5 influenza pandemic caused by an unusually severe and deadly influenza A virus. The victims were often healthy young adults, in contrast to most influenza outbreaks, which predominantly affect juvenile, elderly, or otherwise-weakened patients. Other examples of viruses capable of causing widespread pandemice include, but are not limited to, avian influenza virus, ebola virus, and vesicular stomatitis virus.
Interfering with virus entry is a novel and attractive therapeutic strategy to control virus infection. Proof of principle of this approach has come from peptidic HIV inhibitor enfuvirtide. While confirming the therapeutic benefit of entry inhibitors for the treatment of viral infections, enfuvirtide has also highlighted potential problems of peptidic antivirals. Large heptad repeat-derived peptides, like enfuvirtide, are costly to manufacture, and poor absorption from the gastrointestinal tract necessitates i.v. delivery.
Influenza Virus
The expanding geographic distribution of the avian influenza A (H5N1) virus has put more humans at risk of infection and absence of pre-existing immunity to these viruses in the human population has raised the concern of a new influenza pandemic. (Trampuz et al., Mayo Clin Proc., 79: 523-530 (2004)). In addition, the virus has crossed the species barrier to cause numerous human fatalities in certain regions of Asia and Europe since 1997. (Beigel et al., 2005 supra). Currently there is no effective vaccine against this virus for humans. (Cox et al., Topley & Wilson's Microbiology and Microbial Infections, Collier L, Balows A, Sussman M., eds., London, pp. 634-698 (2005); Kemble, G., and H. Greenberg, Vaccine, 21: 1789-1795 (2003)).
The family Orthomyxoviridae comprises influenza A, B, and C viruses, and Thogoto- and Isavirus. (Cox et al., 2005, supra; Lamb, R. A., and R. M. Krug, Fields Virology, Fourth ed., vol. 1, pp. 1487-1531, Knipe, D. M., Howley, P. M., eds., Lippincott Williams and Wilkins Publishers, Philadelphia (2001)). Influenza pandemics in humans are caused by influenza A viruses. Influenza A viruses contain 8 single-stranded, negative-sense viral RNAs (vRNAs) that encode 10-11 proteins. (Lamb, R. A., 2001, supra; Wright, P. F., and R. G. Webster, Fields Virology, Fourth ed., vol. 1, p. 1533-1579, Knipe, D. M., Howley, P. M., eds., Lippincott Williams and Wilkins Publishers, Philadelphia (2001)).
Influenza A virus contains two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (FIG. 1). Based on the antigenicity of the HA and NA proteins, 16 different HA subtypes (H1-H16) and 9 different NA subtypes (N-1-N9) of influenza A viruses have been identified. Of these, only a limited number of virus subtypes circulate in humans (i.e., H1-H3, and N1, N2). (Lamb, R. A., 2001, supra; Wright, P. F., 2001, supra).
HA is the major viral antigen and mediates receptor-binding and membrane fusion activities, while NA is a receptor-destroying enzyme which releases the viral particles from the cell surface. (Lamb, R. A., 2001, supra; Wright, P. F., 2001, supra). The prototypic HA is synthesized as a single polypeptide and subsequently cleaved into HA1 and HA2 subunits. HA cleavage is required for infectivity, because it generates the hydrophobic N-terminus of HA2, which mediates fusion between the viral envelope and the cell membrane. (Steinhauer, D. A., Virology, 258: 1-20 (1999); Skehel, J. J. and D. C. Wiley, Annu. Rev. Biochem., 69: 531-569 (2000); Lamb, R. A., 2001, supra; Wright, P. F., 2001, supra).
Several steps in the HA-mediated entry process are attractive targets for new anti-influenza therapeutics:
(a) Attachment of HA to its sialic acid receptors: The receptor-binding site of HA is a pocket located on each subunit at the distal globular part of HA1, and binds to cell surface sialic acid residues in a multivalent attachment process. The residues (Y98, W153, H183, E190, L194) forming the pocket are largely conserved among all subtypes of influenza. (Skehel, J. J., 2000, supra).
Therefore, inhibitors can be developed that will block binding of the virus to cells by either binding to receptor binding sites or preventing the interaction through some other mechanism. Several high molecular weight polymers, such as polyphenol and lignin, have been reported to inhibit binding of the virus to the cell membrane. (Sakagami et al., Sieb. Et Zucc. In Vivo, 6: 491-496 (1992); Mochalova et al., Antiviral Research, 23: 179-190 (1994); Sidwell et al., Chemotherapy, 40: 42-50 (1994)).
(b) HA-mediated virus-cell fusion: Influenza virus enters its host cell by receptor-mediated endocytosis, followed by acid-activated membrane fusion in endosomes. The low pH environment in the endosomes is required to trigger the transition of HA from the non-fusogenic to the fusogenic conformation. This conformational change relocates the fusion peptide segment from the amino-terminus of HA2 to the tip of the molecule. Following this conformational change, the fusion peptides fuse the viral envelope with the endosomal membrane. Inhibition ofendosomal H+-ATPase, that blocks the acidification of endosomes, strongly inhibits the replication of influenza virus in MDCK cells. (Hernandez et al., Annu. Rev. Cell Dev. Biol., 12: 627-661 (1996)). However, endosomal H+-ATPase activity is not a virus-specific target and interfering with H+-ATPase activity may lead to undesirable toxic side effects;
(c) The fusogenic trimer-of-hairpins structure: Class I fusion proteins can assume three distinct conformational states, the (i) nonfusogenic native structure, in which the fusion peptide is buried within the trimeric protein; (ii) transient prehairpin intermediate, in which the N-terminal fusion peptide is extended to penetrate the host-cell target membrane; and (iii) fusogenic trimer-of-hairpins structure, in which the C- and N-terminal heptad repeat peptides (HR-C and HR-N) are associated in a six-helix-bundle conformation, is critical for fusion. (Hernandez et al., 1996, supra; Dutch et al., Biosci. Rep., 20: 597-612 (2000); Eckert, D. and Kim P., Annu. Rev. Biochem., 70: 777-810 (2001)). The fusion trimer-of-hairpins structure is maintained through protein-protein interactions. At the C-terminal of the HR-N trimer surface, a deep groove exists that opens into a cavity, forming a hydrophobic pocket recognized as a potential binding site for small molecule inhibitors. Targeting the conserved receptor binding domain and fusogenic trimer-of-hairpins structure will lessen the probability of changes conferring drug-resistance.
Influenza Pandemics
Influenza pandemics are caused by “antigenic shift”, i.e., the introduction of new HA (or new HA and NA) subtypes into the human population. (Cox et al., Annu. Rev. Med., 51: 407-421 (2000)). The lack of prior exposure to the new HA (or HA and NA) subtypes creates a population that is immunologically naive to the “antigenic shift” variants, resulting in extremely high infection rates and rapid spread worldwide. In the 20th century, a total of three major pandemics have occurred: The 1918/1919 ‘Spanish influenza’ is the most devastating infectious disease on record. An estimated 20-50 million people died worldwide and life expectancy in the US was reduced by 10 years. (Johnson et al., Bull. Hist. Med., 76: 105-115 (2000)). The causative agent of ‘Spanish influenza’ was H1N1 influenza A virus, which may have been introduced into human populations from an avian species. (Gamblin et al., Science, 303: 1838-1842 (2004); Reid et al., Nature Rev. Microbiol, 2: 909-914 (2004); Stevens et al., Science, 303: 1866-1870 (2004)). In 1957 and 1968, the ‘Asian influenza’ and ‘Hong Kong influenza’ killed an estimated 70,000 and 33,800 people in the US, respectively. (Johnson et al., 2002, supra). These two pandemic influenza virus strains also arose from reassortment of human and avian strains. (Scholtissek et al., Virology, 87: 13-20 (1978); Kawaoka et al., J. Virol., 63: 4603-4608 (1989); Nakajima et al., Nature, 274: 334-339 (1978)).
Outbreaks of Highly Pathogenic H5N1 Avian Influenza Viruses
Although highly pathogenic H5N1 viruses have not yet caused a human pandemic, their continued transmission to humans and high mortality rate in humans have made the development of therapies to these viruses a priority. The first transmission of highly pathogenic H5N1 avian influenza viruses to humans occurred in Hong Kong in 1997 when 6 of the 18 individuals infected succumbed to the infection. Since 2003, highly pathogenic H5N1 avian influenza viruses have become prevalent in Southeast Asia and endemic in poultry in some countries in this region. (Fauci, A. S., Cell, 124: 665-670 (2006)). More than 4200 outbreaks have been reported in Asian, African, and European countries resulting in the death or slaughter of >100 million poultry. (Beigel et al., 2005, supra). Close contact between humans and poultry in rural areas of these regions likely facilitate virus transmission to humans. 236 human infections with 138 fatalities have been reported in 9 different countries. (Beigel et al., 2005, supra). Furthermore, the appearance of oseltamivir (NA inhibitor) resistant strains of H5N1 indicates that novel therapeutic treatments are urgently needed. (Le et al., 2005, supra).
Options for Pandemic Control
An ideal way to combat the H5N1 avian influenza virus or any emerging or re-emerging influenza virus in humans is to inhibit or at least reduce the likelihood of interspecies transfer, and this requires a comprehensive, multifaceted approach. Currently, vaccination is the proven effective strategy for protection against influenza infection. However, its efficacy during a pandemic will be limited as a ‘pandemic vaccine’ cannot be developed in advance against new emerging strain(s). (Hayden, F. G., 2004, supra). The current inactivated trivalent vaccine does not provide protection against the H5 and H7 avian influenza strains. (Cox et al., 2005); Kemble, G., and H. Greenberg, 2003). Moreover, at this point we cannot predict whether the currently circulating H5N1 will be the next pandemic strain. In addition, the vaccine production capabilities will be strained during a pandemic by the need to immunize a vast number of individuals worldwide in a short period of time. Therefore, antiviral drugs are the first line of medical intervention.
Current anti-influenza drugs, oseltamivir and zanamivir efficiently block the NA activity of the 2004 H5N1 viruses in vitro indicating their effectiveness in influenza chemotherapy and prophylaxis against H5N1 virus infection. (Ward et al., 2005, supra; Mase et al., Virology, 332: 167-176 (2005); Gubareva et al., Lancet, 355: 827-835 (2000)). However, recent isolation of resistant mutants against these compounds has emphasized the urgent need for development of new antivirals. (Le et al., 2005, supra). Development of a new antiviral that will block the conserved receptor binding site or fusion domain of HA is a promising approach and will complement other mechanistic approaches.
Ebola Virus
Ebola viruses cause acute, lethal hemorrhagic fevers for which no vaccines or treatments currently exist. Ebola virus GP is a type I transmembrane glycoprotein. Comparisons of the predicted amino acid sequences for the GPs of the different Ebola virus strains show conservation of amino acids in the amino-terminal and carboxy-terminal regions with a highly variable region in the middle of the protein. (Feldmann et al., Virus Res., 24: 1-19 (1992)). The GP of Ebola viruses are highly glycosylated and contain both N-linked and O-linked carbohydrates that contribute up to 50% of the molecular weight of the protein. Most of the glycosylation sites are found in the central variable region of GP.
The membrane-anchored glycoprotein is the only viral protein known to be on the surfaces of virions and infected cells, and is presumed to be responsible for receptor binding and fusion of the virus with host cells. As a result, Ebola glycoprotein may be an important target preventing viral entry into a host cell. Development of a preventative treatment for Ebola virus is confounded by the observation that Ebola glycoprotein occurs in several forms. The transmembrane glycoprotein of Ebola viruses is unusual in that it is encoded in two open reading frames. Expression of glycoprotein occurs when the 2 reading frames are connected by transcriptional or translational editing. (Sanchez et al., Proc. Natl. Acad. Sci. USA 93: 3602-3607 (1996); Volchkov et al., Virology, 214: 421-430, (1995)). The unedited GP mRNA produces a non-structural secreted glycoprotein (sGP) that is synthesized in large amounts early during the course of infection. (Volchkov et al., 1995, supra; Sanchez et al., 1996, supra; Sanchez et al., J. Infect. Dis. 179 (suppl. 1, S164, (1999). Following editing, the virion-associated transmembrane glycoprotein is proteolytically processed into 2 disulfide-linked products. (Sanchez et al., J. Virol., 72: 6442-6447 (1998)). The amino-terminal product is referred to as GP1 (140 kDa) and the carboxy-terminal cleavage product is referred to as GP2 (26 kDa). GP1 and membrane-bound GP, covalently associate to form a monomer of the GP spike found on the surfaces of virions. (Volchkov et al., Proc. Natl. Acad. Sci. USA 95: 5762 (1998); Sanchez et al., J. Virol., 72: 6442 (1998)). GP1 is also released from infected cells in a soluble form. (Volchkov. et al., Virology, 245: 110 (1998)). sGP and GP1 are identical in their first 295 N-terminal amino acids, whereas the remaining 69 C-terminal amino acids of sGP and 206 amino acids of GP1 are encoded by different reading frames. Development of a new antiviral that will prevent receptor binding and fusion of the virus with a host cell is a promising approach for a vaccine against ebola and any virus with a similar mechanism for entering, i.e., infecting a host cell.
Vesicular Stomatitis Virus (VSV)
Vesicular Stomatitis Virus (VSV) is a non-segmented negative-stranded RNA virus and belongs to the family Rhabdoviridae, genus Vesiculovirus. VSV causes a contagious disease in horses, cattle, pigs, sheep and goats, characterized by vesicular lesions on the tongue, oral mucosa and udder and is transmitted by arthropod vectors. The prominent clinical presentation of vesicular stomatitis is the development of vesicles and ulcers in the oral cavity and, less frequently, on the teats and coronary bands. Mortality rates are typically very low, but production suffers because affected animals lose weight and may develop lameness or mastitis. The most significant concern with vesicular stomatitis is that, in cattle and pigs, it is clinically indistinguishable from foot and mouth disease and swine vesicular disease. Consequently, outbreaks of vesicular stomatitis lead to rapid imposition of international quarantines and shutoff of trade of animals and animals products.
There is also a public health concern because humans can be infected, Patterson, W. C., et al., J. Am. Vet. Med. Ass., 133, 57 (1958), and the virus may be spread by insect vectors, Ferris et al., J. Infect. Dis., 96, 184 (1955), Tesh et al., Science, 175, 1477 (1972).
VSV contains a single negative strand of ribonucleic acids (RNA), which encodes 5 messenger RNA's (mRNA's) and its eleven kb genome has five genes which encode five structural proteins of the virus: the nucleocapsid protein (N), which is required in stoichiometric amounts for encapsidation of the replicated RNA; the phosphoprotein (P), which is a cofactor of the RNA-dependent RNA polymerase (L); the matrix protein (M) and the attachment glycoprotein (G) (e.g., see Gallione et al., 1981 J. Virol., 39:529-535; Rose and Gallione, 1981, J. Virol., 39:519-528; U.S. Pat. No. 6,033,886; U.S. Pat. No. 6,168,943).
The vesicular stomatitis virus envelope protein G binds to the host cell surface to initiate infection. The viral envelope protein participates in virus binding to and/or entry of the infectious virus into a host cell.
Interfering with virus entry is a novel and attractive therapeutic strategy to control virus infection. Proof of principle of this approach has come from peptidic HIV inhibitor enfuvirtide. While confirming the therapeutic benefit of entry inhibitors for the treatment of viral infections, enfuvirtide has also highlighted potential problems of peptidic antivirals. Large heptad repeat-derived peptides, like enfuvirtide, are costly to manufacture, and poor absorption from the gastrointestinal tract necessitates i.v. delivery.